METHODOLOGIES FOR THE APPLICATION OF MONOBROMOBIMANE TO THE SIMULTANEOUS ANALYSIS OF SOLUBLE AND PROTEIN THIOL COMPONENTS OF BIOLOGICAL-SYSTEMS
METHODOLOGIES FOR THE APPLICATION OF MONOBROMOBIMANE TO THE SIMULTANEOUS ANALYSIS OF SOLUBLE AND PROTEIN THIOL COMPONENTS OF BIOLOGICAL-SYSTEMS
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DOI：
10.1016/0165-022x(86)90102-8
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发表时间：
1986-11-01
影响因子：
--
通讯作者：
MOLDEUS, P
中科院分区：
文献类型：
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作者：
COTGREAVE, IA;MOLDEUS, P
介绍了一系列测定生物体系中低分子量和蛋白质硫醇氧化还原状态的简单方法。基于集中使用的monobromobimane，我们描述了一个标准的原位衍生化过程，同时导致最大限度地恢复自由，减少低分子量和溴化imane访问蛋白硫醇作为其相应的bimane加合物从完整的生物系统。试验系统包括分离和培养的细胞、组织匀浆和体液（如血浆）。半胱氨酸和谷胱甘肽的双满加合物的定量通过反相高效液相色谱法实现，而蛋白质硫醇的相应加合物的定量通过蛋白质沉淀后的荧光光谱法实现。描述了定量估计的完整验证数据。此外，我们已经将这些程序耦合到生物蛋白样品的预衍生化变性处理，以定量天然蛋白质样品中溴化丙啶无法接触的蛋白质硫醇池。我们还将这些程序与二硫苏糖醇研究中的生物系统的prederivatization还原结合起来，同时使氧化的低分子硫醇和氧化的蛋白质硫醇可以与monobromobimane进行衍生化。因此，我们已经获得了半胱氨酸和谷胱甘肽的定量测定存在于混合二硫化物与蛋白质和可溶性低分子量二硫化物和蛋白质内二硫化物的估计在一些测试生物系统。
A series of simple methodologies for the determination of the redox status of low molecular weight and protein thiols in biological systems is described. Based centrally upon the use of monobromobimane, we describe a standard in situ derivatization procedure simultaneously resulting in maximal recovery of both free, reduced low molecular weight and bromobimane accessible protein thiols as their corresponding bimane adducts from intact biological systems. Test systems include isolated and cultured cells, tissue homogenates and body fluids such as blood plasma. Quantitation of the bimane adducts of cysteine and glutathione is achieved by reversed phase high performance liquid chromatography, whereas quantitation of the corresponding adducts of protein thiols is achieved by fluorescence spectroscopy following protein precipitation. Full validation data for quantitative estimates are described. Additionally, we have coupled these procedures to prederivatisation denaturation treatments of biological protein samples in order to quantitate pools of protein thiols which are inaccessible to bromobimane in samples of native protein. We have also coupled these procedures with prederivatisation reductions of biological systems under study with dithiothreitol, rendering simultaneously both oxidized low molecular thiols and oxidized protein thiols accessible to derivatisation with monobromobimane. Thus, we have obtained quantitative determinations of cysteine and glutathione present in mixed disulfides with protein and in soluble low molecular weight disulfides and estimates of intraprotein disulfides in a number of test biological systems.